This proposal is directed at elucidation of the biochemical mechanisms involved in differentiation of human promyelocytic leukemia cells (HL-60). Treatment of HL-60 cells with phorbol esters result in differentiation to a macrophage-like cell. The biochemical mechanisms by which phorbol esters induce differentiation in this leukemic cell line remains unclear. It has been hypothesized that the phorbol ester induced differentiation is dependent on the activation of a calcium/phospholipid-dependent protein kinase (C-kinase). Phorbol esters stimulate a number of early biochemical changes which have been felt to be a result of activation of C-kinase. We have recently described a compound, Bryostatin, which activates HL-60 C-kinase both in vitro and in whole cells, but when applied to HL-60 cells in culture does not induce differentiation. In addition, Bryostatin when added concomitantly with phorbol esters prevents the differentiation event. This data suggests that activation of C-kinase is not totally sufficient for HL-60 differentiation. This proposal will focus on the role of C-kinase in phorbol ester induced differentiation of HL-60 cells. Bryostatin will be an important tool to aid in this analysis. We shall first examine whether the early biochemical changes induced after phorbol ester treatment, phosphorylation of specific proteins, phospholipid synthesis and sodium/hydrogen ion flux, occur as a result of C-kinase activation. By examining the effects of Bryostatin, we will establish whether or not these events occur as part of a chain of events initiated by activation of C-kinase or are independent events. Using Bryostatin, will next examine if changes in the nucleus occurring with differentiation are a direct result of C-kinase binding and activation of phosphorylation of specific nuclear substrates. Finally using counterflow centrifugal elutriation to synchronize HL-60 cells, we will examine whether phorbol ester effects on biochemical changes and differentiation are limited to a specific phase in the cell cycle. Studies described in this proposal will aid in the understanding of the role of phorbol esters in HL-60 differentiation. This information could then be used to help understand the block in differentiation in human promyelocytic leukemia.